The present invention relates to a specific binding analysis method for performing a qualitative or quantitative analysis of an analyte in a sample.
With the recent expansion of medical care in households and communities as well as increase of clinical examinations requiring high urgency, there is an increasing demand for the development of a specific binding analysis method which can be performed even by persons other than the experts of the clinical examination, in a rapid, simple and accurate manner.
Many methods are known as the conventional specific binding analyses, which include immunoassay utilizing an antigen-antibody reaction, receptor assay employing a receptor and nucleic acid probe assay employing the hybridization of complementary nucleic acid sequences. Because of their high specificity, these specific binding analysis methods are being frequently used in the clinical examinations and in many other fields.
More specific examples of the immunoassay include chromatography. In chromatography, a liquid sample is brought into contact with a matrix comprising, for example, a porous carrier or a fine particle-packed carrier in each of which a specific binding substance is insolubilized (immobilized). Then, the presence or absence of an analyte in the sample is analyzed by utilizing a phenomenon in which the liquid sample flows out along the matrix by permeating force caused by capillarity (see, e.g., Japanese Patent Nos. 2504923 and 2667793, Japanese Examined Patent Publication No. Hei 7-78503, Japanese Unexamined Patent Publication Nos. Hei 10-73592 and Hei 8-240591).
More specifically, a specific binding substance, which is labeled with a labeling material freely detectable by naked eyes or with an optical method, is specifically bound to an analyte. The specific binding substance specifically bound to the analyte is then bound to a binding material immobilized on the matrix. Finally, the presence or absence of the analyte in the sample is analyzed, according to the labeled amount of the specific binding substance insolubilized on the matrix.
The carrier comprising the matrix used for such chromatography has a large surface area where a great amount of a specific binding substance can be immobilized, so that the collision between reacting molecules, which may cause a specific binding reaction, occurs with a higher frequency as compared to the reaction in a liquid phase. Accordingly, the above-described chromatography is advantageous from the viewpoint of the measurement sensitivity and the measurement time.
In the conventional chromatography described above, it is necessary to employ, as a matrix material, a water absorbing material in which a liquid sample can develop and move by capillarity. Examples of the water absorbing material include a glass fiber filter paper, cellulose film, nitrocellulose film and nylon film, each of which is porous material having a pore size of approximately 1 to 50 μm.
Among them, nitrocellulose is superior, because it is capable of binding to a large amount of protein such as an antibody without being previously sensitized. Moreover, nitrocellulose is commercially available in various pore sizes, so that the use of nitrocellulose makes it is possible to select the flow rate of a sample.
However, the pore sizes and surface hydrophilicity of a matrix material as describe above, comprising a fibrous material, is difficult to be controlled with a high degree of reproducibility in manufacturing. The mean value and distribution of the pore size, as well as the surface hydrophilicity of the fibrous material, have a significant influence on a velocity at which a sample develops and move, that is, a flow rate of the sample. Since a time during which a specific binding reaction occurs greatly depends on the flow rate of the sample, a measured value fluctuates with a change in the flow rate.
In other words, a measured value is extremely sensitive to the properties of the matrix material, and therefore, the measurement accuracy depends on the manufacturing accuracy of the matrix material.
Moreover, it is difficult to improve the manufacturing accuracy of the matrix material to such an extent that a sufficient accuracy is ensured in a quantitative measurement. Accordingly, it has been required to perform the step of screening the matrix material, resulting in a problem of increased cost. Since the pore size range and manufacturing accuracy of the matrix material are limited, there is also a limitation on the range of the sample flow rates to be selected.